Luis Salgado 11/7/12 Wed 9am KT & JW

Lab #4: Protein Functionality: Solubility

I. PURPOSE/ OBJECTIVE The objective is to find the protein solubility for whey and casein solutions by recording the absorbance of samples to calculate the protein concentration.

II. INTRODUCTION Food proteins posses a variety of functional properties like foaming, texture, elasticity, or viscosity. Controlling these functional properties leads to making steady food products. For example, whey protein has come a long way from being a simple cheese waste-product to an ordinary food additive. Its all due to the manipulation of its functional properties with improved methods of processing and modifications. Changing the structure of a protein changes its functionality. The structure may be changed when its environment is changed, whether the protein is denatured or not, or the presence of additional food ingredients. However, actual experimentation is needed to analyze a proteins characteristics in foodstuff. This is where food scientists test for functional application of food proteins by obtaining the protein solutions solubility. A proteins solubility depends on its range of isoelectric point (pI). The isoelectric point is the pH in which a proteins charges are neutral. When a proteins pI is reached, it leaves it vulnerable for structural changes, and by extension also its functional properties.

III.PROCEDURE The procedure followed for the experiment is found in Principles of Food Composition, Laboratory Manual, FS&T 101A Fall 2012 Experiment 4, Protein Functionality: Solubility and

Foam Formation, pages 37-48. Modifications included different schemes for the series of BSA dilutions.

IV. DATA/ RESULTS Table 1a: This table shows the concentration and absorbance (ABS) data for the whey protein samples standard curve made with BSA, where [BSA] = [protein] and ABS is at 595 nm.
Tube 1 2 3 4 5 BSA (L) 0 12.5 25 50 100 Water (L) 100 87.5 75 50 0 [BSA] (mg/ mL) 0 0.1 0.2 0.4 0.8 Absorbance 0 0.065 0.156 0.366 0.786

Bradford Assay Standard Curve for Whey Protein Sample

0.8 0.6 ABS @ 595 nm 0.4 0.2 0 -0.2 y = 1.0025x - 0.0262 R! = 0.9968

0.2

0.4 [Protein] (mg/mL)

0.6

0.8

[BSA] (mg/mL)

Best Fit Line

Figure 1a: This figure is the Bradford Assay Standard Curve for the whey protein samples with the equation of the best fit line.

Table 1b: This table shows the concentration and absorbance (ABS) data for the casein protein samples standard curve made with BSA, where [BSA] = [protein] and ABS is at 595 nm.
Tube 1 2 3 4 5 BSA (L) Water (L) [BSA] (mg/ mL) 0 0.1 0.2 0.4 0.8 ABS

0 12.5 25 50 100

100 87.5 75 50 0

0 0.104 0.285 0.532 0.985

Bradford Assay Standard Curve for Casein Sample

1.500 y = 1.241x + 0.0089 R! = 0.9957

ABS @ 595 nm

1.125

0.750

0.375

0.2

0.4 [Protein] (mg/mL)

0.6

0.8

[BSA] (mg/mL)

Best Fit Line

Figure 1b: This figure is the Bradford Assay Standard Curve for the casein protein samples with the equation of the best fit line.

Table 2: Absorbance Data for the heated (H) and non-heated (NH) whey and casein protein samples at the designated pH conditions.
pH Absorbance (ABS) Whey Protein Sample Casein Protein Sample NH H NH H

pH 2.67 3.52 4.4 5.5 6.65 7.49 8.33 0.335 0.438 0.410 0.486 0.690 0.686 0.490

Absorbance (ABS) 0.440 0.502 0.5 0.480 0.27 0.042 0.59 0.368 0.845 0.478 0.815 0.522 0.518 0.5

0.396 0.514 0.042 0.305 0.482 0.534 0.528

Table 3: The table shows the calculated % solubility of the two proteins, H and NH, as a function of pH
pH Whey Protein Sample NH H 36 46 44 51 71 71 51 % Solubility Casein Protein Sample NH H 46 80 52 76 30 5.2 61 58 87 76 84 82 54 80

2.67 3.52 4.4 5.5 6.65 7.49 8.33

62 82 5.2 48 76 84 84

Heated vs Non-Heated Whey Protein Solubility

90.0

67.5 % Solubility

45.0

22.5

2.67

3.52

4.4

5.5 pH

6.65

7.49

8.33

NH % Solubility

H % Solubility

Figure 2: The graph measures the % solubility of H and NH whey protein samples as a function of pH.

Heated vs Non-Heated Casein Protein Solubility

90.0

67.5 % Solubility

45.0

22.5

2.67

3.52

4.4

5.5 pH

6.65

7.49

8.33

NH % Solubility

H % Solubility

Figure 3: The graph measures the % solubility of H and NH casein protein samples as a function of pH.

V. CALCULATIONS Calculations for the final whey protein concentration: Best fit line equation for the whey Bradford Assay standard curve: y = 1.0025x - 0.0262 Where y = ABS; x = [protein] @ pH 2.5, 0.335 = 1.0025x - 0.0262 x = (0.335 + 0.0262) / 1.0025 = 0.36 mg/ mL of whey protein

Calculations for the % solubility: Original [whey] is 0.1% or 0.1g/ 100mL so: (0.36 mg/ mL) x (1g/1000mg) x (100mL) = 0.036g/mL

% Solubility = (Final Protein Concentration / Original Protein Concentration) x 100 % Solubility = (0.036 g/ 100 mL / 0.1 g/ 100mL) x 100 = 36% Whey Solubility

Calculations for the final casein protein concentration: Best fit line equation for the casein Bradford Assay standard curve: y = 1.241x - 0.0089 Where y = ABS; x = [protein] @ pH 2.5, 0.502 = 1.241x + 0.0089 x = (0.502 - 0.0089) / 1.241 = .40 mg/ mL of casein

Calculations for the % solubility: Original [casein] is 0.05% or 0.05g/ 100mL so; (0.40 mg/ mL) x (1g/1000mg) x (100 mL) = 0.040/ 100mL % Solubility = (Final Protein Concentration / Original Protein Concentration) x 100 % Solubility = (0.04 g/ 100mL / 0.05 g/ 100mL) x 100 = 80% Casein Solubility

VI. DISCUSSION It seemed only right to have two standard curves because of shared data of the two proteins. Due to logistical reasons, one group measured and recorded a Bradford Assay for their standard curve and the other group also did their own. Calculations were made with respective standard curve readings for the purpose of consistency. Nevertheless, the use of a standard curve is the same. The Bradford Assay standard curve was utilized to ultimately give the proteins concentration through absorbance readings. Using Beers Law, we can rearrange it to find the concentration of the absorbing species (the protein). Obtaining the best fit line equation from the standard curve gives the level of absorbance with its rate(slope). We can find a proteins concentration by dividing the absorbance by the slope. Although its suppose to be a standard curve, there is room for error. For the sake of consistency, the data for the whey protein was evaluated against its experimenters calculated standard curve and the data for the casein protein was evaluated against its own (and different) experimenters calculated standard curve. There was plenty of room for error during the

spectrophotometer readings. There were small off-readings given by the machine so it had to be re-calibrated/re-zeroed. Another possibility for error lies in the denaturing of the protein with excessive stirring from the stirring rod. This unfolding of the protein couldve exposed inhibited amino acids that now would be able to aggregate through hydrogen bonding or hydrophobic interactions with the environment. The isoelectric point (pI) of a protein is reached when theres minimum solubility because its charge is neutral, hence cannot react with the polar environment of water. The more polar a proteins surface is, the more soluble it tends to be. If the pH passes the pI, then the charge is negative and vise versa. Data from figures 2 and 3 indicate that the greatest solubility for both proteins is between pH 6.5-7.5. This is logical because it seems that the isoelectric point for both proteins lies between 4.4-4.6 and as pH begins to increase, it lowers the amount of Hydrogen protons, resulting in a more polar protein surface. Figure 3 shows the casein solubility with a severe, sharp plunge at its pI at pH of 4.4-4.6 and its because it precipitates around this pH. This allows for hydrogen bonding to occur between the hydrophilic groups on the protein itself, changing the proteins conformation. The whey protein shows a greater solubility after pH 4.6 from Figure 2. This is explained by the wheys hydrophilic and hydrophobic amino acid mixture composition. After its pI is passed, the whey protein sample solution has the ability for its hydrophilic amino acids to interact with the polar environment. These interactions give the whey protein a higher % solubility. The whey protein is affected by heat at a higher degree than the casein protein. Figure 2 shows that the Heated samples give a curve with a greater solubility % and noticeable pI where as the Non-Heated samples show a curve that has a small increase for solubility after its vague pI. The wheys globular structure unfolds with heat and so denatures to an insoluble substance during its pI stage. As the pH begins to pick up again, the hydrophilic sites can bond to the polar water. The casein protein samples did not show the same results that whey demonstrated with the Heated modification. Both curves are alike and almost parallel. Its solubility % peak has only a 2% difference between the Heated vs Non-Heated with Heated having the higher solubility%.

VII.CONCLUSION

This experiment compared the solubility of two proteins (whey and casein) under varying conditions (pH levels and heated vs non-heated) by manipulating Beers Law and using a standard curve so that together, would make it possible to calculate the protein concentration. A spectrophotometer was utilized for the Absorbance readings at a wavelength of 595 nm with recalibration for every other reading. Three parts were done in this experiment to fulfill this comparison, which were protein solubility at different pH levels established, measuring a standard curve for reference, and protein assay. Plotting the graphs confirms the proteins isoelectric point and establishes a sense of understanding of protein solubility with changing pH levels as well as heat influence. A proteins solubility is experimentally determined and is explained by the proteins interaction with water as it reaches a neutral charge (pI) and then passes it with higher pH levels.

VIII.QUESTIONS 1. Using your data, identify the pH at which whey and casein proteins are least soluble. Describe the condition of the protein and this pH which causes this point of minimum solubility. Whey is least soluble at pH of 4.4 and casein is least soluble at pH of 4.4 as well. Whey protein is least soluble at this pH because the globular structure with heat unfolds and denatures to an insoluble structure because of disulfide bridges. Casein is at its most insoluble state at pH4.4 because the negatively charged GMP gets neutralized by lowering the pH to its pI, therefore removing repulsive electrostatic forces between the micelles allowing them to aggregate via hydrophobic interactions.

2. At their respective point of minimum solubility, contrast the degree of solubility between whey and casein proteins for both heated and non-heated protein solutions. Discuss how molecular structures influence the degree of solubility for each type of protein. Whey proteins minimum solubility is amplified when the sample is heated than when its not. However, Casein proteins minimum solubility isnt affected by heat as much as the whey. Nevertheless, it still has a lower minimum solubility point than whey. Molecular structures influence the degree of solubility for whey in that it has a mixture of hydrophobic and

hydrophilic amino acids. This explains why it has a higher minimum solubility than casein; the hydrophilic amino acids are soluble where as the casein does not have any chance for hydrophilic amino acids interacting with the polar water. The casein proteins are more affected by the change in pH level than in change in temperature.